Application of lactobacillus johnsonii YH1136 inactivated thallus in preparation of products for relieving depression, anxiety and sleep disorders

Abstract

The invention provides application of lactobacillus johnsonii YH1136 inactivated thalli in preparation of products for relieving depression, anxiety and sleep disorders, and relates to the technical field of microorganisms. The inactivated thallus is obtained by carrying out heat treatment on live thallus of lactobacillus johnsonii YH1136 with the preservation number of CCTCC NO: M 20221116 at 60 to 68 DEG C for 30 to 90 minutes. Animal experiments show that the inactivated thallus can significantly improve depression-like behaviors, anxiety-like behaviors and sleep disorders of CUMS model mice, and the effect of the inactivated thallus is equivalent to that of viable bacteria and positive drug fluoxetine, and is significantly superior to that of 100 DEG C traditional high-temperature inactivated thallus. The action mechanism is related to inhibition of HPA hyperfunction and up-regulation of hippocampal brain-derived neurotrophic factors and prefrontal cortex 5-hydroxytryptamine levels. The inactivated thallus provided by the invention is high in stability, good in safety and suitable for developing related drugs.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and particularly relates to application of inactivated Lactobacillus johnsonii YH1136 in preparation of a product for relieving depression, anxiety and sleep disorders. BACKGROUND

[0002] Depression, anxiety and sleep disorders are common mental health problems that seriously affect the quality of human life, especially in the modern society with fast pace and high pressure, the incidence continues to rise. Depression is characterized by persistent low mood and decreased interest, often accompanied by anxiety, sleep disorders and other comorbidities, which brings heavy burden to patients, families and society. At present, the first-line antidepressants in clinical mainly based on the monoamine neurotransmitter hypothesis, which play a therapeutic effect by increasing the levels of 5-hydroxytryptamine (5-HT) and norepinephrine, but have problems such as slow onset, limited effectiveness, obvious side effects and large individual differences, which seriously affect the treatment compliance and quality of life of patients. Therefore, it is of great clinical and social significance to develop alternative or auxiliary therapies with novel mechanism of action, high safety and small side effects.

[0003] In recent years, with the in-depth development of the "gut-brain axis" theory, the bidirectional regulation relationship between gut microbiota and central nervous system has become increasingly clear. A large number of studies have shown that specific probiotics can improve depression, anxiety-like behavior and sleep quality by regulating gut flora balance, reducing inflammatory response, enhancing intestinal barrier function, and affecting neuroendocrine and immune pathways. Although live probiotics show good prospects in regulating mood, they still face many challenges in practical application: the survival rate of live bacterial preparations is easily affected by processing, storage and gastrointestinal environment, and the stability is poor; for individuals with low immunity or damaged intestinal barrier, live bacteria have potential infection or migration risks; in addition, the metabolic activity of live bacteria may cause abdominal distension, flatulence and other digestive discomfort. Therefore, it is a technical problem to be solved in the field to develop a microbial preparation with high stability, good safety, convenient storage and transportation, and effective regulation of mood and sleep.

[0004] As a new type of microbial preparation, inactivated bacteria retains the structure and bioactivity of bacterial components, avoids the reproduction and colonization risks of live bacteria, and has better processing stability, storage tolerance and use safety, which has shown application potential in regulating immunity and improving intestinal health. However, the current application research of specific inactivated bacteria in relieving depression, anxiety and sleep disorders is still limited, especially the lack of inactivated strains with strong pertinence and clear mechanism of action and their preparation process. SUMMARY

[0005] Therefore, the application aims to provide a use of Lactobacillus johnsonii YH1136 inactivated bacteria in preparation of a product for relieving depression, anxiety and sleep disorders, the inactivated bacteria being prepared through a specific heat treatment process, which not only maintains the biological active components of the bacteria, but also has excellent stability and safety, thereby providing a new solution for developing a new, safe and stable product for emotion and sleep regulation.

[0006] To achieve the above-mentioned application purposes, the application provides the following technical solutions. The application provides an application of Lactobacillus johnsonii YH1136 inactivated bacteria in preparation of a product for relieving depression, anxiety and / or sleep disorders.

[0007] Preferably, the Lactobacillus johnsonii YH1136 inactivated bacteria is obtained by heat treatment inactivation of Lactobacillus johnsonii YH1136 live bacteria with a preservation number of CCTCC NO: M 20221116.

[0008] More preferably, the temperature of the heat treatment is 60-68 DEG C, and the treatment time is 30-90 min.

[0009] Preferably, the product is a pharmaceutical composition.

[0010] The application also provides a composition for relieving depression, anxiety and / or sleep disorders, and an active component of the composition is obtained by heat treatment inactivation of Lactobacillus johnsonii YH1136 live bacteria with a preservation number of CCTCC NO: M 20221116.

[0011] Preferably, the temperature of the heat treatment is 60-68 DEG C, and the treatment time is 30-90 min.

[0012] Preferably, the composition is a pharmaceutical composition, and further comprises a pharmaceutically acceptable carrier.

[0013] More preferably, the pharmaceutically acceptable carrier comprises one or more of a diluent, a binder, a disintegrant, a lubricant, a flavoring agent and a stabilizer.

[0014] The application also provides a method for preparing the composition, comprising the following steps: (1) culturing Lactobacillus johnsonii YH1136 to obtain live bacteria; (2) preparing the live bacteria into a bacterial suspension; (3) treating the bacterial suspension at a temperature of 60-68 DEG C for 30-90 min to inactivate and obtain Lactobacillus johnsonii YH1136 inactivated bacteria; (4) formulating the Lactobacillus johnsonii YH1136 inactivated bacteria with a pharmaceutically acceptable carrier into the composition.

[0015] Preferably, the initial concentration of viable bacteria in the bacterial suspension is 1x10 9 CFU / mL to 5x10 10 CFU / mL.

[0016] Compared with the prior art, the present application has the following beneficial effects: (1) The Lactobacillus johnsonii YH1136 inactivated bacteria provided by the present application is prepared by a mild heat treatment process at 60-68℃, which can completely inactivate while retaining the integrity of the bacterial structure and key bioactive components to the greatest extent, solving the problems of poor stability of live bacterial preparations and potential safety risks.

[0017] (2) The inactivated bacteria described in the present application exhibit comparable antidepressant, anxiolytic and sleep-improving effects to live bacteria and the clinical positive drug fluoxetine in a chronic unpredictable mild stress (CUMS) animal model, and its mechanism is closely related to the regulation of the hypothalamic-pituitary-adrenal axis and the up-regulation of hippocampal brain-derived neurotrophic factor (BDNF) and prefrontal cortex 5-hydroxytryptamine (5-HT) levels.

[0018] (3) The 60-68℃ mild heat treatment process used in the present application maximizes the retention of the biological activity of the bacteria under the premise of complete inactivation. Experiments show that the traditional 100℃ high-temperature inactivated bacteria are significantly inferior to the present application in various behavioral and biochemical indicators; and prolonging the heat treatment time or increasing the temperature will also lead to a decrease in activity. This proves that the process conditions of the present application have achieved the best balance between inactivation completeness and activity retention, solving the problems of poor stability of live bacterial preparations, high storage requirements and potential safety risks.

[0019] Therefore, the present application provides a new technical solution and product form for developing stable, safe and effective microbial preparations for relieving mental and psychological disorders and sleep problems.

[0020] Biological preservation certificate The Lactobacillus johnsonii YH1136 described in the present application is classified and named as Lactobacillus johnsonii YH1136, the preservation unit is China Center for Type Culture Collection, the preservation unit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the preservation number is CCTCC NO: M20221116, and the preservation date is July 15, 2022. DETAILED DESCRIPTION

[0021] The present application provides an application of Lactobacillus johnsonii YH1136 inactivated bacteria in the preparation of products for relieving depression, anxiety and / or sleep disorders.

[0022] In the present application, the Lactobacillus johnsonii YH1136 inactivated bacteria are obtained by heat treatment of the live bacteria of Lactobacillus johnsonii YH1136 with the preservation number CCTCC NO: M20221116. In specific embodiments of the present application, the Lactobacillus johnsonii YH1136 inactivated bacteria are prepared by the following steps: first, the live bacteria of Lactobacillus johnsonii YH1136 with the preservation number CCTCC NO: M20221116 are activated and cultured; then, the bacteria are collected by centrifugation and resuspended with sterile normal saline to prepare a bacterial suspension; finally, the bacterial suspension is subjected to heat treatment at a temperature of 60-68°C for 30-90 minutes to completely inactivate the bacteria, thereby obtaining the Lactobacillus johnsonii YH1136 inactivated bacteria. This preparation method can ensure that the bacteria are inactivated while their structural integrity and relevant bioactive components are maximally retained.

[0023] In the present application, the temperature of heat treatment is preferably 60-68°C, and the treatment time is preferably 30-90 minutes. In specific embodiments of the present application, the prepared bacterial suspension of Lactobacillus johnsonii YH1136 is placed in a constant-temperature water bath at 65°C for 60 minutes. This condition has been experimentally verified to maximize the structural integrity of the bacterial cells while ensuring complete inactivation of the bacteria and effectively retaining the bioactive components such as specific surface proteins or cell wall polysaccharides related to mood and sleep regulation. In the present application, if the temperature is lower than 60°C, the bacteria may not be completely inactivated or the required time may be too long; if the temperature is higher than 68°C, the bacterial proteins may be excessively denatured and the structure may be severely damaged, resulting in loss of key active substances. The treatment time and temperature work synergistically, and a treatment time of 30-90 minutes is sufficient to achieve the best balance between complete inactivation and activity retention at the preferred temperature. Other combinations within this range, such as 62°C for 80 minutes or 68°C for 30 minutes, also fall within the scope of the present application and can be verified by routine experiments for complete inactivation and effectiveness.

[0024] In the present application, the depression, anxiety and / or sleep disorder is preferably induced by chronic unpredictable mild stress. In specific embodiments of the present application, a chronic unpredictable mild stress (CUMS) animal model is established to simulate the core symptoms of human depression, anxiety and related sleep disorders, and the intervention effect of the product of the present application is evaluated on this model.

[0025] In the present application, the product is preferably a pharmaceutical composition. As a preferred embodiment, the inactivated bacteria as an active pharmaceutical ingredient need to be combined with one or more pharmaceutically acceptable carriers or excipients to prepare a dosage form suitable for administration. Specifically, it includes but is not limited to oral solid preparations or oral liquid preparations.

[0026] The present application also provides a composition for relieving depression, anxiety and / or sleep disorders, wherein the active ingredient of the composition is obtained from heat-treated inactivated Lactobacillus johnsonii YH1136 with the CCTCC NO: M 20221116.

[0027] In the present application, the temperature of the heat treatment is preferably 60-68℃, and the treatment time is preferably 30-90min.

[0028] In the present application, the composition is a pharmaceutical composition, and further comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes one or more of diluents, binders, disintegrants, lubricants, flavoring agents and stabilizers.

[0029] The present application also provides a method for preparing the composition, which preferably comprises the following steps: (1) culturing Lactobacillus johnsonii YH1136 to obtain live bacteria; (2) preparing the live bacteria into a bacterial suspension; (3) inactivating the bacteria by treating the bacterial suspension at a temperature of 60-68℃ for 30-90min to obtain inactivated Lactobacillus johnsonii YH1136; (4) formulating the inactivated Lactobacillus johnsonii YH1136 with a pharmaceutically acceptable carrier into the composition.

[0030] The method for preparing the composition provided by the present application has a clear and repeatable specific embodiment, and aims to obtain a stable, safe and specific bioactive end product.

[0031] In the specific embodiment of the present application, starting from the original preserved Lactobacillus johnsonii YH1136 with the CCTCC NO: M 20221116, streak activation is performed on a suitable solid culture medium, and anaerobic culture is performed at 37℃ for 24-48 hours. Then, the culture is expanded: a single colony is picked and inoculated into a liquid culture medium for seed liquid culture. Preferably, the inoculation amount is 1%-3% (v / v), and the seed liquid is cultured at 37℃ under anaerobic conditions for 12-18 hours to obtain a high-activity seed liquid. As a preferred embodiment, the seed liquid is transferred to a fermenter or a larger volume of liquid culture medium with an inoculation amount of 2%-5%, and large-scale culture is performed under controlled temperature (37℃), pH and anaerobic environment until the stable late growth phase is reached (usually cultured for 16-24 hours), at which time the biomass and activity of the bacteria are in an ideal state. After the culture is completed, the wet bacteria are separated and collected by centrifugation at 4℃ and 5000-8000×g for 10-20 minutes, and then washed with sterile physiological saline or buffer to remove the culture medium ingredients.

[0032] In the present application, the collected bacterial cells are resuspended in physiological saline or phosphate buffered saline (PBS). The bacterial cells are uniformly dispersed by vortexing or gentle homogenization to form a uniform bacterial suspension. The concentration of the bacterial suspension is adjusted to 1 x 10 9 CFU / mL to 5 x 10 10 CFU / mL. This concentration range is conducive to uniform and efficient subsequent heat treatment and ensures the dosage of active ingredients in the final product.

[0033] In the present application, the prepared bacterial suspension is placed in a device that can accurately control the temperature, and the bacterial suspension is subjected to constant temperature treatment at a temperature range of 60°C to 68°C for 30 minutes to 90 minutes. One preferred embodiment is to accurately treat at 65°C for 60 minutes. After heat treatment, immediately take samples and spread on nutrient agar plates, and culture under suitable conditions for at least 48 hours to confirm that there is no colony growth to ensure that the bacterial cells are completely inactivated. In a specific embodiment of the present application, the obtained inactivated bacterial suspension can be used directly or the inactivated bacterial cells are collected by centrifugation. To further improve the stability, the inactivated bacterial slurry can be preferably freeze-dried to prepare inactivated bacterial cell freeze-dried powder for storage and subsequent preparation.

[0034] In the present application, the inactivated bacterial cells are preferably formulated with a pharmaceutically acceptable carrier into the composition. As a preferred embodiment, when the composition is a pharmaceutical composition, the dosage form of the final preparation can be determined according to the type of target product and the administration route, such as tablets, capsules, granules, powders or oral solutions. The obtained Lactobacillus johnsonii YH1136 inactivated bacterial cells are mixed as active ingredients with one or more pharmaceutically acceptable carriers or excipients. These carriers / excipients include but are not limited to diluents (such as starch, microcrystalline cellulose), binders, disintegrants, lubricants (such as magnesium stearate), flavoring agents or stabilizers. For solid oral preparations (tablets, capsules), the mixture can be directly compressed into tablets or filled into capsule shells. For granules or powders, granulation, drying and sizing can be performed before packaging. For oral solutions, the inactivated bacterial cells can be uniformly dispersed or dissolved in an aqueous medium containing suspending agents, preservatives and pH adjusters, and then filled.

[0035] In the present application, the initial concentration of viable bacterial cells in the bacterial suspension is preferably 1 x 10 9 CFU / mL to 5 x 10 10 CFU / mL. In a specific embodiment of the present application, the concentration of the bacterial suspension is accurately adjusted to the target range by direct dilution or centrifugal concentration, with conventional microbiological counting techniques such as hemocytometer, photometer or plate colony counting method. As a preferred embodiment, the initial concentration of the bacterial suspension is adjusted to 5 x 10 10CFU / mL. This concentration can obtain high yield and high activity of inactivated bacteria after subsequent heat treatment at 65℃ / 60min, and is easy to control the process.

[0036] The technical solutions provided by the present application are described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the present application.

[0037] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.

[0038] Example 1 Preparation of Lactobacillus johnsonii YH1136 inactivated bacteria The Lactobacillus johnsonii YH1136 described in this example is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20221116. The taxonomic name of the Lactobacillus johnsonii YH1136 strain is Lactobacillus johnsonii YH1136.

[0039] Take the live bacteria of Lactobacillus johnsonii YH1136 with the preservation number of CCTCC NO: M 20221116, inoculate in MRS liquid medium, and anaerobically culture at 37℃ for 20h to obtain live bacteria culture. Centrifuge the culture at 5000r / min for 10min at 4℃, collect the bacteria, resuspend with sterile normal saline, and adjust the concentration of the bacterial suspension to 5×10 10 CFU / mL. Take 10mL of bacterial suspension and place it in a constant temperature water bath, heat inactivate at 65℃ for 60min. After inactivation, take samples and spread on MRS plates, culture at 37℃ for 48h, confirm no live bacteria growth, obtain Lactobacillus johnsonii YH1136 inactivated bacteria suspension, and store at 4℃ for standby.

[0040] Comparative Example 1 Take the live bacteria of Lactobacillus johnsonii YH1136 with the preservation number of CCTCC NO: M 20221116, inoculate in MRS liquid medium, and anaerobically culture at 37℃ for 20h to obtain live bacteria culture. Centrifuge the culture to collect the bacteria, resuspend with sterile normal saline, and adjust the concentration of the bacterial suspension to 5×10 10 CFU / mL, without heat treatment, store at 4℃ for standby.

[0041] Comparative Example 2 Industrial high temperature instantaneous treatment Take 10mL of bacterial suspension and place it in a constant temperature water bath, heat inactivate at 100℃ for 15min in boiling water bath. After inactivation, take samples and spread on MRS plates, culture at 37℃ for 48h, confirm no live bacteria growth, obtain Lactobacillus johnsonii YH1136 inactivated bacteria suspension, and store at 4℃ for standby.

[0042] Comparative Example 3 long-time heat treatment Take 10 mL of bacterial suspension and place it in a constant temperature water bath, and continuously treat it at 65°C for 120 min. After the treatment, immediately place the sample in an ice bath to cool to room temperature, to obtain the inactivated Lactobacillus johnsonii YH1136 bacterial suspension, which is stored at 4°C for later use.

[0043] Comparative Example 4 high-temperature long-time heat treatment Take 10 mL of bacterial suspension and place it in a constant temperature water bath, and continuously treat it at 70°C for 120 min. After the treatment, immediately place the sample in an ice bath to cool to room temperature, to obtain the inactivated Lactobacillus johnsonii YH1136 bacterial suspension, which is stored at 4°C for later use.

[0044] Comparative Example 5 pasteurization type heat treatment Take 10 mL of bacterial suspension and place it in a constant temperature water bath, and continuously treat it at 80°C for 30 min. After the treatment, immediately place the sample in an ice bath to cool to room temperature, to obtain the inactivated Lactobacillus johnsonii YH1136 bacterial suspension, which is stored at 4°C for later use.

[0045] Comparative Example 6 ultrasonic treatment Take 10 mL of bacterial suspension and place it in a sample tube of an ultrasonic cell disruptor, and place the sample tube in an ice water bath to prevent overheating during the treatment. Set the instrument power to 200 W, the frequency to 20 kHz, and the pulse mode (work for 2 s, interval for 1 s), and the total treatment time is 20 min. After the treatment, obtain the inactivated Lactobacillus johnsonii YH1136 bacterial suspension, which is stored at 4°C for later use.

[0046] Take 100 μL of the bacterial suspension after treatment in each of the comparative examples and Example 1, and evenly spread it on MRS solid agar plates. At the same time, to improve the detection sensitivity, take 1 mL of each sample, concentrate it with sterile normal saline, and then spread it on the plates. Place all the plates in an anaerobic environment at 37°C, and continuously culture them for 7 days. The results show that no colonies are formed on the plates of Example 1 and Comparative Examples 2-5, indicating that the bacterial cells are completely inactivated under these heat treatment conditions. The plates of Comparative Example 6 (ultrasonic treatment) have very few sporadic colonies appearing after the late stage of the culture, indicating that the inactivation is not complete under this condition, and there are still a very small amount of live bacteria remaining.

[0047] At the same time, scanning electron microscopy of the bacterial sample prepared in Example 1 shows that the bacterial cells obtained by the inactivation method of the present application have an intact morphology and clear surface structure.

[0048] Example 2 Effect verification of Lactobacillus johnsonii YH1136 inactivated bacterial cells on relieving depression, anxiety and sleep disorders of a chronic unpredictable mild stress (CUMS) mouse model 1. Experimental animals and grouping 7-8 weeks old male C57BL / 6J mice, weighing 20-22 g, were randomly divided into 7 groups, 10 in each group: ① Normal control group (Control): no stress was applied, and an equal volume of normal saline was administered by gavage every day; ② CUMS model group (CUMS): CUMS stimulation was applied, and an equal volume of normal saline was administered by gavage every day; ③ Positive drug group (Flu): CUMS stimulation was applied, and fluoxetine (10 mg / kg) was administered by gavage every day; ④ Inactivated bacteria low-dose group (YH1136-L): CUMS stimulation was applied, and the inactivated bacteria suspension of Example 1 (equivalent to 5×10 9 CFU / mL) was administered by gavage every day; ⑤ Inactivated bacteria high-dose group (YH1136-H): CUMS stimulation was applied, and the inactivated bacteria suspension of Example 1 (equivalent to 1×10 10 CFU / mL) was administered by gavage every day; ⑥ Live bacteria control group (YH1136-LIVE): CUMS stimulation was applied, and the live bacteria suspension of Comparative Example 1 (equivalent to 1×10 10 CFU / mL) was administered by gavage every day; ⑦ Industrial high-temperature instantaneous treatment bacteria group (YH1136-100): CUMS stimulation was applied, and the inactivated bacteria suspension of Comparative Example 2 (equivalent to 1×10 10 CFU / mL) was administered by gavage every day.

[0049] ⑧ Long-time heat treatment bacteria group (65℃, 120min): CUMS stimulation was applied, and the live bacteria suspension of Comparative Example 3 (equivalent to 1×10 10 CFU / mL) was administered by gavage every day; ⑨ High-temperature long-time heat treatment bacteria group (70℃, 120min): CUMS stimulation was applied, and the inactivated bacteria suspension of Comparative Example 4 (equivalent to 1×10 10 CFU / mL) was administered by gavage every day; ⑩ Pasteurization type heat treatment bacteria group (80℃, 30min): CUMS stimulation was applied, and the inactivated bacteria suspension of Comparative Example 5 (equivalent to 1×10 10 CFU / mL) was administered by gavage every day; Ultrasonic treatment bacteria group: CUMS stimulation was applied, and the inactivated bacteria suspension of Comparative Example 6 (equivalent to 1×10 10 CFU / mL) was administered by gavage every day.

[0050] 2. CUMS procedure The following one or more stress stimuli were randomly applied daily for 5 weeks according to the literature method (Willner et al., 1992): ① 24 h water deprivation; ② 24 h food deprivation; ③ 12 h wet bedding; ④ 12 h tilt cage at 45°; ⑤ 30 min restraint stress; ⑥ light reversal at night; ⑦ 1 h noise stress.

[0051] 3. Behavioral tests (1) Sucrose preference test The test was performed before CUMS, at the end of the 3rd week and the 5th week. The mice were singly housed and deprived of water and food for 12 h, and then two pre-weighed water bottles were provided simultaneously (one containing 1% sucrose solution and the other containing pure water). After 1 h, the positions of the two bottles were exchanged, and the test was ended after another 1 h. The sucrose preference rate (%) = sucrose consumption / (sucrose consumption + pure water consumption) x 100%.

[0052] Table 1 Results of sucrose preference test at the end of the 5th week in each group

[0053] Note: Compared with the model group, ###P<0.001, ##P<0.01, #P<0.05; compared with the inactivated bacteria high-dose group, .

[0054] According to the results in Table 1, the preference rate of the CUMS model group was significantly lower than that of the normal group, indicating that the model was successfully constructed. The sucrose preference rate (76.8%) of the inactivated high-dose group (YH1136-H) of the present application was at the same high level as that of the positive drug fluoxetine group (78.5%) and the live bacteria control group (79.1%), and there was no statistical difference, indicating that the bacteria inactivated by the process of the present application completely retained the biological activity of relieving the loss of pleasure. In comparison, the industrial high-temperature treatment (100℃) group improved weakly (55.3%), only with marginal significant difference from the model group; and prolonging the heat treatment time (65℃ / 2h) or increasing the temperature (70℃ / 2h, 80℃ / 30min) all led to a gradient decline in effect, and there was a significant difference from the present application group.

[0055] (2) Tail suspension test and forced swimming test Tail suspension test (TST) and forced swimming test (FST) are the most commonly used analysis methods for studying the depressive-like behavior of rodents, and are often used as a detection method for screening potential antidepressants.

[0056] ① Tail suspension test The depression-like behavior of mice was detected by TST. In the tail suspension test (55 cm x 15 cm x 11.5 cm), the mouse tail was fixed on a hook 30 cm above the ground, and a camera was placed horizontally to the mouse suspension device. Before the experiment, the animal tail was suspended to make the mouse in an inverted body position, and the head should be kept a certain distance from the bottom of the suspension box. At the beginning of the experiment, the mouse was fixed on a hook, and the immobile time of the mouse within 4 min was observed and recorded by a video imaging system.

[0057] 2. Forced swimming test FST is a test method for evaluating the depression-like behavior of mice. In a quiet environment, the mouse was placed in a transparent water tank (30 cm x 11 cm) containing about 20 cm deep water, and the water temperature was maintained at 25 ± 1℃. After each mouse test, the water in the water tank was replaced with clean water to remove the mouse's excrement and odor to avoid interference with subsequent mouse tests. The behavior of the mouse was observed and recorded by a video imaging system, and the total recording time was 6 min. The immobile time of the mouse within the last 4 min was statistically analyzed.

[0058] Table 2 Comparison of immobile time in tail suspension and forced swimming test of each group

[0059] Note: Compared with the model group, ###P<0.001, ##P<0.01, #P<0.05; compared with the inactivated bacteria high-dose group, .

[0060] According to the results in Table 2, in the two tests, the immobile time of the CUMS model group was significantly prolonged, indicating that the depression-like behavior modeling was successful. Importantly, the high-dose bacteria (YH1136-H) inactivated by the process of the application (65℃, 60min) had no statistical difference in immobile time with the live bacteria control group (YH1136-LIVE) and the clinical positive drug fluoxetine group, proving that the inactivation treatment completely retains the core activity of the bacteria against behavior despair. At the same time, although the low-dose group (YH1136-L) showed significant improvement, its effect was significantly weaker than that of the high-dose group in all indicators, which clearly demonstrated the dose dependence of the effect of the application. Further process comparison results showed that prolonging the heat treatment time (65℃ / 2h) led to a significant decrease in effect; increasing the treatment temperature (70℃ / 2h, 80℃ / 30min) or using ultrasonic inactivation led to a more obvious decline in effect; and traditional industrial high temperature (100℃) treatment almost completely lost the activity of the bacteria. These two groups of mutually corroborating data strongly indicated that the inactivation step of the application effectively maintained the balance between complete inactivation and activity retention, so that the inactivated bacteria produced by the application achieved the same biological effect as the live bacteria preparation in terms of anti-depression-like behavior.

[0061] (3) Elevated plus maze test The anxiety-like behavior of mice was detected by EPM. The elevated plus maze (35 cm x 5 cm x 10 cm) was composed of two open arms, two closed arms and a central region. At the end of the 5th week, the mice were placed in the center of the elevated plus maze with their heads facing the open arms, and their activities were observed and recorded for 5 min using a video imaging system. After each mouse test, the elevated plus maze was cleaned with 75% alcohol to remove mouse excrement and odors to avoid interference with subsequent mouse tests. The number of times and the time spent in the open arms were recorded within 5 min. Open arm time ratio (%) = open arm time / total test time x 100%.

[0062] Table 3 Open arm time ratio of each group in the elevated plus maze test

[0063] Note: Compared with the model group ②, ###P<0.001, ##P<0.01, #P<0.05; compared with the high-dose inactivated bacteria group ⑤, .

[0064] The elevated plus maze test was used to evaluate anxiety-like behavior, and a decrease in open arm exploration time is a typical manifestation of anxiety. According to the results in Table 3, the CUMS model group showed extremely significant anxiety-like behavior in this indicator. The open arm exploration time of the high-dose inactivated bacteria group of the present application returned to normal levels, and its effect was no different from that of the positive drug and live bacteria control group, proving that the inactivation process of the present application also completely retains the anti-anxiety biological activity of the bacteria. In sharp contrast, the effects of other inactivation methods all showed different degrees of decline. Traditional high-temperature treatment at 100°C only produced weak improvement. More importantly, at 65°C, doubling the treatment time (2h) led to a significant decline in effect; and when the temperature was raised to 70°C or short-time treatment at 80°C was used, the effect declined even more obviously. This indicates that the functional components that regulate anxiety-like behavior may be more sensitive to heat, or the maintenance of their active conformation depends on more precise thermodynamic conditions.

[0065] (4) Sleep-wake cycle monitoring The non-invasive mouse activity recording system was used to continuously monitor the diurnal activity rhythm and sleep time of mice for 72h at the end of the 5th week.

[0066] Table 4 Monitoring results of 24-hour total sleep time of each group

[0067] Note: Compared with the model group ②, ###P<0.001, ##P<0.01, #P<0.05; compared with the high-dose inactivated bacteria group ⑤, .

[0068] Chronic stress often leads to sleep disorders, which are evaluated by monitoring total sleep time. According to the results in Table 4, the sleep time of the CUMS model group is significantly shortened. The inactivated bacterial cell intervention of the present application can effectively reverse this phenomenon, restore the total sleep time to the normal level without difference from the positive drug group and the live bacteria group, and prove the clear effect of improving stress-related sleep disorders. However, this sleep improvement function is also highly dependent on the inactivation process. The industrial high temperature (100℃) and 80℃ short-time pasteurization treatment cannot significantly increase the sleep time. Even at 65℃, the improvement effect is significantly weaker than that of the inactivated bacterial cell group of the present application for 1 hour, and the ultrasonic treatment effect is also limited. These data collectively show that the bacterial cell component or structure mediating sleep regulation needs to be optimally preserved under the specific mild heat treatment conditions described in the present application.

[0069] Example 3 Effect of Lactobacillus johnsonii YH1136 inactivated bacterial cells on serum biochemical indicators of mice 1. Serum Corticosterone (CORT) level The mice in each group in Example 2 were euthanized after the behavioral test, and an appropriate amount of mouse blood was taken and centrifuged at 4℃ at a speed of 3000 rpm / min for 10 minutes. The supernatant was taken, and the CORT level was detected by using an ELISA kit.

[0070] Table 5 Detection results of serum corticosterone (CORT) levels of mice in each group

[0071] Note: Compared with the ② model group, ###P<0.001, ##P<0.01, #P<0.05; compared with the ⑤ inactivated bacterial cell high-dose group, .

[0072] Serum corticosterone (CORT) is a key marker of the activity of the hypothalamic-pituitary-adrenal (HPA) axis, and its chronic elevation is an important neuroendocrine feature of depression. According to the results in Table 5, the inactivated bacterial cell high-dose group of the present application can significantly reduce the serum CORT level, and its effect is completely comparable to that of the positive drug fluoxetine and live bacteria treatment. It is revealed that one of the core mechanisms of the anti-depression effect of the product of the present application is to inhibit excessive stress response by regulating the gut-brain axis. The preservation of this regulation function is extremely sensitive to heat treatment. The traditional 100℃ inactivated bacterial cells cannot reduce the CORT level. Even if the treatment temperature is increased to 65℃ for a long time (2h), the effect is significantly weaker than that of the standard process (1h) of the present application; when the treatment temperature is increased to 70℃ or 80℃, the function of the bacterial cells in regulating the HPA axis is almost lost. This shows that the bacterial-related molecular pattern mediating this down-regulation has clear heat sensitivity.

[0073] 2. Hippocampus tissue brain-derived neurotrophic factor (BDNF) content After the behavior test, the mouse brain tissue was taken, the hippocampus tissue was separated on ice, and the pre-cooled PBS buffer was added according to a certain mass-volume ratio for ice bath homogenization. The supernatant was obtained by centrifugation, the content was detected by using a mouse BDNF ELISA kit, the protein concentration of the supernatant was detected by using a BCA method, and the final result was expressed as pg / mg protein.

[0074] Table 6 Detection results of hippocampus tissue brain-derived neurotrophic factor (BDNF) content of mice in each group

[0075] Note: compared with the model group ②, ###P<0.001, ##P<0.01, #P<0.05; compared with the inactivated bacteria high-dose group ⑤, .

[0076] According to the results in Table 6, it can be seen that the CUMS model in the experiment caused a significant decrease in the hippocampus BDNF content. The inactivated bacteria intervention of the application can effectively up-regulate the hippocampus BDNF level, so that it returns to the normal range without difference from the positive drug and live bacteria treatment. This result is crucial, which elucidates the potential mechanism of the long-term antidepressant effect and the promotion of neural plasticity of the product from the molecular and cellular levels. However, the bacteria treated at 100℃ has a significantly weakened ability to enhance BDNF. The further comparative experiment shows that the function is significantly reduced when the treatment time is doubled at 65℃; the function is lost more seriously when the treatment temperature is increased to 70℃ or 80℃. This proves that the maintenance of the biological activity conformation of the bacteria signal molecules or components that can stimulate the host hippocampus tissue to produce BDNF depends on an accurate thermodynamic balance.

[0077] 3. Prefrontal cortex 5-hydroxytryptamine (5-HT) content The mouse brain tissue was taken, and the prefrontal lobe was separated on ice. The PBS buffer was added according to a certain mass-volume ratio, homogenized, centrifuged to obtain the supernatant, and the content was detected by using a 5-HT ELISA kit, and the result was expressed as pg / mg protein.

[0078] Table 7 Detection results of prefrontal cortex 5-hydroxytryptamine (5-HT) content of mice in each group

[0079] Note: compared with the model group ②, ###P<0.001, ##P<0.01, #P<0.05; compared with the inactivated bacteria high-dose group ⑤, .

[0080] According to the results of Table 7, the experiment proves that the CUMS model causes the 5-HT content in the brain region to decrease significantly. The inactivated bacteria bodies by the process of the application can effectively improve the effect of 5-HT level, which is completely comparable to the classic SSRI drug fluoxetine and the live bacteria itself. This provides direct evidence for the effect of the product of the application from the level of key neurotransmitters, and reveals that the product of the application may be synergistic or alternative to the traditional drug. The neurotransmitter regulation function shows high specificity to the inactivation conditions. The traditional high temperature (100℃) treatment makes the function of the bacteria bodies basically lost. Under the same temperature (65℃), prolonging the treatment time leads to a significant weakening of the effect; increasing the treatment temperature (70℃, 80℃) causes more serious function degradation. This shows that the active substances of the bacteria bodies responsible for ultimately affecting the central 5-HT system through the gut-brain axis depend on the complete function of the mild heat treatment conditions defined by the application. Therefore, the inactivation method of the application is not only a safe sterilization step, but also a key preparation process for directional retention and optimization of specific neuroregulation functions.

[0081] The above only describes the preferred embodiments of the application, and it should be noted that for those skilled in the art, without departing from the principles of the application, several improvements and refinements can be made, and these improvements and refinements should also be considered within the protection scope of the application.

Claims

1. Application of inactivated Lactobacillus johnsonii YH1136 cells in the preparation of products for relieving depression, anxiety and / or sleep disorders.

2. The application according to claim 1, characterized in that, The inactivated Lactobacillus johnsonii YH1136 cells were obtained by heat treatment inactivation of live Lactobacillus johnsonii YH1136 cells with accession number CCTCC NO:M 20221116.

3. The application according to claim 2, characterized in that, The heat treatment temperature is 60℃-68℃, and the treatment time is 30min-90min.

4. The application according to claim 1, characterized in that, The product is a pharmaceutical composition.

5. A composition for relieving depression, anxiety, and / or sleep disorders, characterized in that, The active ingredient of the composition is obtained by heat treatment inactivation of live Lactobacillus johnsonii YH1136 cells with accession number CCTCC NO:M 20221116.

6. The composition according to claim 5, characterized in that, The heat treatment temperature is 60℃-68℃, and the treatment time is 30min-90min.

7. The composition according to claim 5, characterized in that, The composition is a pharmaceutical composition and also contains a pharmaceutically acceptable carrier.

8. The composition according to claim 7, characterized in that, The pharmaceutically acceptable carriers include one or more of the following: diluents, binders, disintegrants, lubricants, flavoring agents, and stabilizers.

9. A method for preparing the composition according to any one of claims 5-8, characterized in that, Includes the following steps: (1) Culture Lactobacillus johnsonii YH1136 to obtain live cells; (2) Prepare the live bacterial cells into a bacterial suspension; (3) The bacterial suspension was treated at a temperature of 60℃-68℃ for 30min-90min to inactivate and obtain inactivated Lactobacillus johnsonii YH1136 cells; (4) The inactivated Lactobacillus johnsonii YH1136 cells were formulated with a pharmaceutically acceptable carrier to form the composition.

10. The preparation method according to claim 9, characterized in that, The initial concentration of live bacteria in the bacterial suspension was 1×10⁻⁶. 9 CFU / mL up to 5×10 10 CFU / mL.

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